GLCNE CRISPR Activation Plasmid (m): sc-424509-ACT

Mouse Gne encodes glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GLCNE), a bifunctional, rate-limiting enzyme in the de novo sialic acid biosynthetic pathway that converts UDP-GlcNAc to ManNAc-6-phosphate. By controlling cellular CMP-sialic acid pools, GLCNE influences protein and lipid sialylation that shapes glycoprotein maturation in the secretory pathway and modulates cell–cell recognition, adhesion, and receptor signaling at the plasma membrane. Altered Gne activity and sialylation homeostasis are linked to defects in muscle physiology and broader glycosylation-associated stress responses that impact inflammatory signaling and extracellular matrix interactions. These biology features make Gne a useful target for studying sialylation-dependent regulation of membrane proteins, trafficking, and glycoconjugate composition in mouse model systems.

GLCNE CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Gne expression without altering the underlying DNA sequence.

GLCNE CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Gne locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the Gne transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GLCNE expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Gne locus and enabling the study of GLCNE-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GLCNE pathway restoration in tumor cells with silenced or reduced Gne expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.